Dynamics of Structural and Functional Changes in ... - Springer Link

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Bulletin of Experimental Biology and Medicine, No. 2, 2001 EXPERIMENTAL BIOLOGY

Dynamics of Structural and Functional Changes in Hepatocyte Mitochondria of Senescence-Accelerated OXYS Rats N. G. Kolosova*++, S. V. Aidagulova**, G. I. Nepomnyashchikh**, I. G. Shabalina, and N. I. Shalbueva+ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 132, No. 8, pp. 235-240, August, 2001 Original article submitted June 18, 2001 Electron microscopy showed signs of degenerative dystrophic changes in hepatocytes and structural decompensation of mitochondria in senescence-accelerated OXYS rats in comparison with Wistar rats. These signs were detected in the presence of liver mitochondria dysfunction in OXYS rats: reduced oxygen consumption rate in all metabolic states, respiratory control volume, ADP/O ratio. transmembrane potential, and phosphorylation rate. The revealed disorders in mitochondrial structure and function are the key factors in the pathogenesis of accelerated aging in OXYS rats and visceropathies characteristic of these animals. Key Words: OXYS rats; hepatocyte mitochondria; electron microscopy; energy metabolism of liver mitochondria Rat model of accelerated aging, OXYS rat strain (initially registered as W/SSM) was created at the Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, by selection and inbreeding of Wistar rats sensitive to cataractogenic effect of galactose [6,8]. Apart from early development of cataract, these animals are characterized by decreased life-span, high incidence of malignant tumors, early involution changes in the viscera, including cardiomyopathies [3], and impaired cognitive function [2]. Hyperproduction of oxygen radicals in the liver and heart detected by electron paramagnetic resonance [14] is believed to underlie the pathogenesis of these symptoms; this hyperproduction was the key characteristic for registration of this mouse strain as OXYS [13]. *Institute of Cytology and Genetics, Siberian Division, Russian Academy of Medical Sciences; **Institute of Regional Pathology and Pathomorphology, Siberian Division, Russian Academy of Medical Sciences; +Institute of Biochemistry, Siberian Division, Russian Academy of Medical Sciences; Institute of Physiology, Siberian Division, Russian Academy of Medical Sciences, Novosibirsk. Address for correspondence: pathol@ cyber.ma.nsc.ru. Kolosova N. C.

The most probable causes of intracellular oxidative stress are disorders in the electron transporting chain of mitochondria, characteristic of OXYS rats, which can be detected as early as at the age of 2-3 months and then progress [7]. However chemiluminescent analysis showed that generation of active oxygen forms by enzymatic systems of liver mitochondria in OXYS rats is even lower than in Wistar rats of the corresponding age [4]. These results seem to be unexpected, as the involvement of free radicals in changes leading to cell and tissue aging has been persuasively demonstrated [9,15,16]. Moreover, it is known that imbalance in the generation of active oxygen forms and energy metabolism can be both the cause and result of aging [12]. We investigated structural and functional changes in liver mitochondria of OXYS rats characterized by accelerated aging.

MATERIALS AND METHODS The study was carried out on 23 male OXYS rats aged 4, 12, and 24 months and weighing 287±48, 332±30, 0007-4888/01/1322-0814$25.00 © 2001 Plenum Publishing Corporation

N. G. Kolosova, S. V. Aidagulova, et al.

and 352±23 g, respectively. Control group consisted of 22 Wistar rats of the same ages weighing 340±60, 379±72, and 396±42 g, respectively. The rats were from Breeding Laboratory of the Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences. For light microscopy, the samples were fixed in 10% neutral formalin and treated routinely. Paraffin sections were stained with hematoxylin-eosin in combination with Pearls reaction, by Van Gieson method with poststaining of elastic fibers by Weigert resorcinfuchsin, and periodic acid Schiff reaction was carried out. Liver fragments (no more than 1 mm 2) for electron microscopy were fixed in 4% paraformaldehyde and postfixed in 1% OsO4. After standard treatment for electron microscopy, the tissue was embedded in epon-araldite mixture. Semithin sections were stained with 1% Azur II. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined under a JEM 1010 electron microscope. Biochemical studies were carried out on 4- and 12-month-old rats. Mitochondria were isolated from the liver by standard differential centrifugation in the following medium: 250 mM sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.4); the mitochondria were repeatedly precipitated in the same medium without EDTA. Mitochondrial protein was measured by the Lowry method with BSA as the reference. Oxygen consumption rate was evaluated by polarography at 25oC using a Clarke electrode. The mitochondria were incubated under the following conditions: 100 mM sucrose, 50 mM KCl, 20 mM Tris-HCl (pH 7.4), 5 mM K2PO4, 1 mM MgCl2, 1 mM EGTA. Oxidation substrate: 10 mM succinate with rotenone 2 µg/mg mitochondrial protein. Transmembrane potential (∆Ψ) was measured with a fluorescent probe 4-n(diaminaminostyryl)-1-methylpyridinium (Zonde) [1]. The results were statistically processed using Statsoft software.

RESULTS Light microscopy showed no marked differences in the liver structure between 4-month-old experimental and control rats, except slightly increased glycogen content in centrolobulbar hepatocytes and moderate peripolesis (lymphocyte migration into interhepatocellular spaces) in OXYS rats. Comparative electron microscopy of hepatocyte ultrastructure in 4-monthold OXYS rats (Fig. 1, a) showed a higher α-glycogen content and numerous mitochondria compactly grouped in various parts of the cytoplasm, often near the granular cytoplasmatic reticulum. In comparison with hepatocyte mitochondria of Wistar rats (Fig. 1, b), they were much more abundant, smaller in size, and had more osmiophilic matrix, the structure and density

815 of cristae being similar. These changes indicated mitochondrial proliferation and increase in their surface area. Moreover, hepatocytes of 4-month-old OXYS rats contained free ribosomes and polysomes; sinusoidal and lateral hepatocyte plasmalemma formed numerous microvilli. Generally, this complex of ultrastructural signs corresponded to the juvenile phenotype (probably due to delayed liver maturation or adaptive restructuring of the liver parenchyma). Light microscopy showed that the liver structure in 12-month-old OXYS rats differed from that of Wistar rats of the same age by a higher glycogen content accumulating in the portocentral direction, moderate mononuclear infiltration of portal tracts, Ito cell hyperplasia, and changed tinctorial characteristics of hepatocyte cytoplasm. Electron microscopy showed progressive degenerative processes in the liver parenchyma of OXYS rats, much more advanced than in the control group. The mitochondrial compartment during this period was characterized by polymorphism of its numerous organelles (Fig. 2, a): the mitochondria were enlarged (compared to 4-month-old animals), had clarified matrix and reduced, thinned, and chaotically oriented cristae. Hepatocyte cytoplasm had lipofuchsin granules (markers of cell hypoxia and/or aging), polymorphic residual bodies, and solitary lipid inclusions. Foci of intracellular regeneration were not so numerous as in Wistar rats of the same age. Liver structure of 24-months-old OXYS rats was characterized by moderately expressed dyscomplectation of hepatic trabecules and parenchymatous cell polymorphism due to uneven glycogen content in the lobules, acidophilic and lipid degeneration of periportal and pericentral hepatocytes, devastation of cytoplasmatic matrix in some hepatocytes. Moderate proliferation of biliary ducts, focal mononuclear infiltration, and solitary lymphoid aggregations were seen in portal tracts; central vein fibrosis was observed. The same changes, but less pronounced, were seen in Wistar rats. Electron microscopy showed that the number of mitochondria and glycogen content were decreased in the majority of hepatocytes of 24-month-old OXYS rats; numerous lipofuchsin granules (Fig. 2, b), autophagosomes containing fragments of degenerated mitochondria, and polymorphic residual bodies were seen. Many hepatocytes contained abundant small mitochondria with rare cristae and homogenous moderately electron-dense matrix, sometimes with osmiophilic granules (pathognomonic for hypoxia and intoxication). This indicated heterogeneity of parenchymatous liver cells maintaining the adaptive compensatory potential in these animals surviving until the critical age (maximum life-span of OXYS rats is 24.6±4.3 months

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Bulletin of Experimental Biology and Medicine, No. 2, 2001 EXPERIMENTAL BIOLOGY

a

b Fig. 1. Ultrastructural characteristics of hepatocytes in 4-month-old OXYS (a) and Wistar (b) rats, ×4000. a) hyperplasia of mitochondria and granular cytoplasmatic reticulum; b) scanty mitochondria and elements of cytoplasmatic reticulum.

N. G. Kolosova, S. V. Aidagulova, et al.

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a

b Fig. 2. Fragments of hepatocytes in OXYS rats, ×4000. a) pronounced mitochondrial polymorphism, formation of residual bodies at the age of 12 months; b) signs of mitochondrial destruction and lipofuchsin granules at the age of 24 months.

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Bulletin of Experimental Biology and Medicine, No. 2, 2001 EXPERIMENTAL BIOLOGY

TABLE 1. Functional State of Liver Mitochondria in Wistar and OXYS Rats (M±m) Wistar

OXYS

Parameter 4 months (n=7)

12 months (n=7)

4 months (n=9)

12 months (n=8)

Oxygen consumption rate, ng atom O/min/mg mitochondrial protein metabolic state 3

158.6±22.0

158.8±18.4

147.0±14.6

83.8±8.7*

metabolic state 4

41.6±6.1

49.0±5.0

47.0±6.8

34.70±2.35*

uncoupling state

212.9±20.0

224.0±17.0

212.0±12.5

155.2±12.9*

Respiratory control

4.00±0.43

3.64±0.26

3.4±0.4

2.50±0.33**

ADP/O

1.65±0.09

1.68±0.07

1.50±0.09

1.37±0.12**

202.0±15.0

207.0±24.0

238.0±21.0

122.0±21.0*

194.0±7.8

198.0±1.9

200.0±3.4

187.00±2.12*

Phosphorylation rate, nM ADP/min/mg mitochondrial protein ∆Ψ, mV

Note. *p